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FoxO limits microtubule stability and is itself negatively regulated by microtubule disruption.

Nechipurenko IV, Broihier HT - J. Cell Biol. (2012)

Bottom Line: Indeed, levels of neuronal FoxO were strongly reduced after acute pharmacological MT disruption as well as sustained genetic disruption of the neuronal cytoskeleton.This decrease was independent of the dual leucine zipper kinase-Wallenda pathway and required function of Akt kinase.We present a model wherein FoxO degradation is a component of a stabilizing, protective response to cytoskeletal insult.

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Affiliation: Department of Neurosciences, Case Western Reserve University, Cleveland, OH 44106, USA.

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Acute MT disruption negatively regulates FoxO protein levels. (A) Representative immunoblots showing protein levels of Fasciclin 2 (Fas 2), DVGLUT, and Nkx6 after 30-min Noc treatment. n ≥ 2 blots/treatment/marker. (B, C, F, and G) Representative confocal images of four abdominal segments of L3 larval VNCs stained for FoxO after the indicated drug treatments. Bars, 20 µm. (D and H) Representative immunoblots showing FoxO protein levels in L3 larval CNS lysates after the listed DMSO and Noc application paradigms. (E and I) Quantification of changes in FoxO protein levels expressed as FoxO/GAPDH ratio normalized to DMSO controls after indicated Noc treatments. n ≥ 3 blots (six CNS/treatment/lane). Anterior is up. Error bars show means ± SEM. *, P < 0.05. Molecular masses are given in kilodaltons.
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fig9: Acute MT disruption negatively regulates FoxO protein levels. (A) Representative immunoblots showing protein levels of Fasciclin 2 (Fas 2), DVGLUT, and Nkx6 after 30-min Noc treatment. n ≥ 2 blots/treatment/marker. (B, C, F, and G) Representative confocal images of four abdominal segments of L3 larval VNCs stained for FoxO after the indicated drug treatments. Bars, 20 µm. (D and H) Representative immunoblots showing FoxO protein levels in L3 larval CNS lysates after the listed DMSO and Noc application paradigms. (E and I) Quantification of changes in FoxO protein levels expressed as FoxO/GAPDH ratio normalized to DMSO controls after indicated Noc treatments. n ≥ 3 blots (six CNS/treatment/lane). Anterior is up. Error bars show means ± SEM. *, P < 0.05. Molecular masses are given in kilodaltons.

Mentions: We next investigated whether neuronal FoxO levels are also modulated by acute disruption of the MT cytoskeleton. We destabilized MTs via application of 100 µM nocodazole (Noc) using a published protocol (Massaro et al., 2009). 30-min Noc incubation disrupts dynamic MTs without visibly altering Futsch-positive MT organization (not depicted: Massaro et al., 2009) or levels of the adhesion molecule Fasciclin 2 at the NMJ (Fig. S4). Additionally, we find no changes in levels of the transcription factors Even skipped and Nkx6, Fasciclin 2, and the vesicular glutamate transporter (DVGLUT) after acute Noc treatment (Fig. 9 A and not depicted), arguing that short-term Noc incubation does not globally compromise neuronal differentiation. However, this drug treatment is sufficient to induce the Fos transcription factor (Massaro et al., 2009), suggesting activation of the JNK stress response pathway (Collins et al., 2006; Miller et al., 2009; Xiong et al., 2010). To ask whether FoxO levels are also modulated in this paradigm, we fixed tissue immediately after Noc incubation and analyzed FoxO expression. The intensity of nuclear FoxO in the CNS is reduced after Noc incubation (Fig. 9, B and C). This decrease is mirrored on Western blots (Fig. 9 D), indicating that it does not reflect altered subcellular localization. Quantification of FoxO protein levels demonstrates a twofold reduction after Noc incubation (Fig. 9 E). It is conceivable that the decrease in FoxO protein reflects regulation at the transcriptional level. However, the stability of FoxO proteins (Huang and Tindall, 2011) coupled with the rapid time course of the down-regulation argues that FoxO is actively degraded after cytoskeletal disruption.


FoxO limits microtubule stability and is itself negatively regulated by microtubule disruption.

Nechipurenko IV, Broihier HT - J. Cell Biol. (2012)

Acute MT disruption negatively regulates FoxO protein levels. (A) Representative immunoblots showing protein levels of Fasciclin 2 (Fas 2), DVGLUT, and Nkx6 after 30-min Noc treatment. n ≥ 2 blots/treatment/marker. (B, C, F, and G) Representative confocal images of four abdominal segments of L3 larval VNCs stained for FoxO after the indicated drug treatments. Bars, 20 µm. (D and H) Representative immunoblots showing FoxO protein levels in L3 larval CNS lysates after the listed DMSO and Noc application paradigms. (E and I) Quantification of changes in FoxO protein levels expressed as FoxO/GAPDH ratio normalized to DMSO controls after indicated Noc treatments. n ≥ 3 blots (six CNS/treatment/lane). Anterior is up. Error bars show means ± SEM. *, P < 0.05. Molecular masses are given in kilodaltons.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3275378&req=5

fig9: Acute MT disruption negatively regulates FoxO protein levels. (A) Representative immunoblots showing protein levels of Fasciclin 2 (Fas 2), DVGLUT, and Nkx6 after 30-min Noc treatment. n ≥ 2 blots/treatment/marker. (B, C, F, and G) Representative confocal images of four abdominal segments of L3 larval VNCs stained for FoxO after the indicated drug treatments. Bars, 20 µm. (D and H) Representative immunoblots showing FoxO protein levels in L3 larval CNS lysates after the listed DMSO and Noc application paradigms. (E and I) Quantification of changes in FoxO protein levels expressed as FoxO/GAPDH ratio normalized to DMSO controls after indicated Noc treatments. n ≥ 3 blots (six CNS/treatment/lane). Anterior is up. Error bars show means ± SEM. *, P < 0.05. Molecular masses are given in kilodaltons.
Mentions: We next investigated whether neuronal FoxO levels are also modulated by acute disruption of the MT cytoskeleton. We destabilized MTs via application of 100 µM nocodazole (Noc) using a published protocol (Massaro et al., 2009). 30-min Noc incubation disrupts dynamic MTs without visibly altering Futsch-positive MT organization (not depicted: Massaro et al., 2009) or levels of the adhesion molecule Fasciclin 2 at the NMJ (Fig. S4). Additionally, we find no changes in levels of the transcription factors Even skipped and Nkx6, Fasciclin 2, and the vesicular glutamate transporter (DVGLUT) after acute Noc treatment (Fig. 9 A and not depicted), arguing that short-term Noc incubation does not globally compromise neuronal differentiation. However, this drug treatment is sufficient to induce the Fos transcription factor (Massaro et al., 2009), suggesting activation of the JNK stress response pathway (Collins et al., 2006; Miller et al., 2009; Xiong et al., 2010). To ask whether FoxO levels are also modulated in this paradigm, we fixed tissue immediately after Noc incubation and analyzed FoxO expression. The intensity of nuclear FoxO in the CNS is reduced after Noc incubation (Fig. 9, B and C). This decrease is mirrored on Western blots (Fig. 9 D), indicating that it does not reflect altered subcellular localization. Quantification of FoxO protein levels demonstrates a twofold reduction after Noc incubation (Fig. 9 E). It is conceivable that the decrease in FoxO protein reflects regulation at the transcriptional level. However, the stability of FoxO proteins (Huang and Tindall, 2011) coupled with the rapid time course of the down-regulation argues that FoxO is actively degraded after cytoskeletal disruption.

Bottom Line: Indeed, levels of neuronal FoxO were strongly reduced after acute pharmacological MT disruption as well as sustained genetic disruption of the neuronal cytoskeleton.This decrease was independent of the dual leucine zipper kinase-Wallenda pathway and required function of Akt kinase.We present a model wherein FoxO degradation is a component of a stabilizing, protective response to cytoskeletal insult.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurosciences, Case Western Reserve University, Cleveland, OH 44106, USA.

Show MeSH
Related in: MedlinePlus